What is transfection?

Transfection is a biotechnology technique, and it is used to deliver foreign genetic material (nucleic acid) into the host cell. Suppose transfection is performed in the laboratory. It is called in-vitro transfection. If the transfection is performed or genetic material is directly injected into the organism, it is known as in-vivo transfection. DNA, RNA, siRNA, and miRNA can be delivered into the organism through transfection. The method, condition of an organism, and genetic material condition are directly proportional to the efficacy of the transfection.  

Different animal models are suitable for the transfection experiment. Mouse (rat) is a widely used animal for biological experiments, and scientists have successfully transfected the mouse and observed its efficacy.  

The mouse has become the preferred mammalian model. The genome of the mouse is 85 percent similar to the human genome. A mouse can be cared for easily without any inconvenience, and breeding and studying a mouse is also easy. Even scientists are producing transgenic mice with desirable traits for research purposes. 

Transfection Methods 

Transfection can be performed using various strategies, including virus-mediated methods (biological) and non-viral methods (physical and chemical) such as electroporation, calcium phosphate exposure, and liposomal transfection that allows delivery of cargo molecules across cell membranes without causing irreversible damage to the cell. 

Many scientific fields use transfection as a common technique for cell culture, drug discovery, and development. An in vitro transfection occurs when cargo molecules (such as nucleic acids – DNA or RNA) are delivered into cells in culture (usually cancer cells). In vivo transfection refers to delivering cargo molecules (e.g., siRNA, plasmid DNA, small proteins) to the target tissue.  

Laboratories use commercial transfection products for in vitro and in vivo transfection. These services are provided by biology CRO companies that are GLP certified to conduct these preclinical experiments per FDA guidelines. 

We describe several methods of transfection below. 

Biological Transfection Methods  (virus-mediated methods) 

Transduction, also known as virus-mediated transfection, is the most common method in clinical research. Transfection mediated by viruses is highly efficient and facilitates sustainable transgene expression in vivo due to its viral nature. In humans, retroviruses such as murine leukemia virus (MLV) have been used to establish sustained transgene expression.  

MLV integrates its DNA into the host genome, and its integrated DNA is expressed in the host. It replicates as normally as the host genome. Thus, it divides into daughter cells, allowing sustainable transgene expression. 

Disadvantages of virus-mediated transfection Method 

• Immunogenicity and cytotoxicity are the major drawbacks of virus-mediated transfection. Virus vectors can cause inflammatory reactions and insertional mutations since they randomly integrate into the host genome, which can interfere with tumor suppressor genes, activate oncogenes, or interrupt essential genes.  
• Another disadvantage of this method is that foreign genes have limited space to remain infectious in a virus package. 

As a result, many attempts have been made to develop non-viral methods of transfection even though virus-mediated approaches are highly effective and very convenient. 

Chemical Transfection Methods 

Chemical transfection is widely used because it is easy, inexpensive, and offers various transfection reagents. Transient transfection is typically done to express a gene for a short period, typically a few days. Studies of gene expression, gene silencing, and recombinant proteins are among the research applications. 

Across all cell types, chemical transfection experiments follow the same workflow. Transfection reagents are prepared immediately before transfection, and construct expression is assessed. Cells are plated on a new medium one day after transfection, and the medium is refreshed a few hours after transfection. The transfection protocol must be optimized for high transfection efficiency and non-toxic results. 

Transfection by lipofectamine involves fusing small molecules (liposomes) with cell membranes to release their contents. Typically, liposomes are composed of lipids – but they can also be composed of other types of molecules. The second transfection method is to use cationic polymers, which have a positive charge and bind well to negatively charged nucleic acids. 

 New medicines are discovered and developed using liposome transfection methods, especially in oncology. Methods include targeting specific genes (such as oncogenes) with small molecule-based drugs or silencing genes using RNA interference (RNAi). 

Physical Transfection Methods 

Physical transfection methods such as electroporation and injection of cells (e.g., gene guns) can accomplish nucleic acid delivery but are harsh on cells due to disrupted cellular membranes, which often results in cell death. It is an appropriate option for cells that have traditionally been difficult to transfect. Microinjection, electroporation, and biolistic particle delivery comprise the physical transfection procedure. 

The amount of DNA and the type of cells used in electroporation determine the results. Electroporation is traditionally performed using an electroporation cuvette. Numerous companies offer electroporation systems that can be used with various cell types, including primary, stem, and mammalian cells. 

DNA and RNA can be introduced into individual cells through microinjection, such as in embryonic stem cells. Direct injection of DNA or RNA into the nucleus or cytoplasm is achieved through a microscope and micromanipulator. Typically, this process is lengthy but results in extremely high transfection efficiency. 

Biolistic Method 

The biolistic particle delivery method, or particle bombardment, is another means of transfection. Nucleic acids are delivered with high-velocity microprojectiles by penetrating the membrane of recipient cells. Various nucleic acids have been delivered to cells in the lab and in vivo using this method. 

Initial cost considerations prevented particle bombardment instruments from being used in research applications, so the method was mainly used in agricultural genetics. More recent developments in nanotechnology, however, have revolutionized the use of particle bombardment instruments. A nanoparticle is a synthetic material with less than 100 nanometers particle size. 

An and Jin (2012) found that nanoparticles can attach to DNA and RNA through covalent or noncovalent interactions due to their small size. As an alternative to pronuclear microinjection or viral vectors, these properties have been utilized for creating transgenic animals. This technique is called “magnetofection,” in which iron oxide nanoparticles are used to deliver genes using magnetic fields. 

Experiments and Protocols for Transfection 

Transfection is a very useful and broad technique, but it can also be extremely specific to the transfected cell type. Transfection works on some cells while not on others, so certain transfection reagents are more effective on some cells.  

Transfection reagents enable experiments to function without extensive research because of the extensive variety of cell lines and the nuances associated with each one. Transfection reagents can be developed specifically for specific cell types, thus ensuring maximum efficiency in gene-editing experiments. 

Designing a transfection experiment requires an in-depth understanding of the ultimate goal; stable expression requires a different setup from transient expression, so experimental protocols should be adjusted accordingly. It is possible to outsource transfection experiments if time is a factor since reliable companies can guarantee the development of stable or transient cell lines for research purposes.